The subject matter of the invention are nucleotide analogues, antiviral pro-nucleotides, a use of nucleotide analogues and pharmaceutical composition, a phosphorylating agent for synthesis of nucleotide analogue, and a method of synthesis of nucleotide analogue. More precisely, the invention applies to the new group of nucleotide analogues and their use in partial or complete inhibition of human immunodeficiency virus (HIV).
There were described many masked nucleotide derivatives called pro-nucleotides, which were converted in the living cells into biologically active nucleotides. Among pro-nucleotides investigated for combating viruses, including HIV, the best results which made these compounds good candidates for therapeutic applications, were obtained for the nucleotide derivatives (covered with patents) called pro-nucleotides, in which nucleosides were 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyuridine (ddU), 2′,3′-dideoxyadenosine (ddA), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-2′,3′-didehydrothymidine (d4T), 9-[9(1,3-dihydroxy-2-propoxy)methyl]guanine, acyclovir (ACV), 2′,3′-dideoxycytidine (ddC), 2′,3′-dideoxy-3′-thiacytidine (3TC).
Bis-pivaloyloxymethyl nucleoside phosphotriesters depicted with formula (I),
are called pivaloyloxymethyl (POM) pro-nucleotides [Sastry J. K. et al., Mol. Pharmacol., 1992, 41, 441-445]. Nucleoside bis-pivaloyloxymethyl [R═C(CH3)3] (POM) phosphotriesters are pro-nucleotides which are converted into respective nucleoside phosphates by means of enzymatic hydrolysis of carboxylic ester of the side chain of pro-nucleotide phosphate masking group. Generated in this way hydroxymethylene functionality, rearranged spontaneously and produced unprotected nucleoside phosphate depicted with formula (II) and formaldehyde.
Nucleoside phosphotriesters depicted with formula (III)
represents bis(S-acylthioethyl) (SATE) pro-nucleotides [Johum A., et al., Organomet. Chem., 2005, 690, 2614-2625]. Compound of type (III) represents a whole family of anti-HIV bis-SATE nucleoside phosphotriesters (bis-SATE pro-nucleotides). Conversion of these compounds into unprotected phosphates of type (II) is initiated with enzymes—carboxyesterases, which hydrolyse carboxylic esters of S-acylthioethyl group and generates 2-thioethyl functionality that spontaneously undergoes elimination with formation of nucleosid-5′-yl phosphate of type (II). SATE nucleotides of type (III) are undoubtedly pro-nucleotides, due to they are internalized into cells as such and in the way described above generated awaited unmasked nucleotides of type (II). An anti-HIV potency of bis-SATE phosphotriesters of type (III) was described for derivatives of many nucleoside analogues of known anti-HIV activity. Unfortunately, bis-SATE pronucleotides (III) although disclosed advantageous pharmacokinetic parameters, bear some inconveniences because they are poorly soluble in water and also because the intermediate phosphodiester appeared to be rather poor substrate for enzymes hydrolyzing carboxylic esters due to close vicinity of phosphate anion (after removing of the first SATE group). There are known SATE derivatives which partially by-passed above inconveniences. There are known combinations of the phosphotriester SATE group with other different type of phosphate masking group of pro-nucleotides e. g. phenyl or phosphoramidate type ones.
The next group are bis(dithioethyl)nucleoside phosphotriesters—dithioethyl pro-nucleotides (DTE) [Gosselin G. et al., Acta Biochem. Polon., 1996, 43(1), 195-208] having the formula (IV)
The DTE groups mask the phosphate moieties in pro-nucleotides (IV), and are removed analogously as the SATE but with an aid of reductase enzymes that cleave disulfide bonds yielding a 2-thioethyl phosphodiester group, which undergoes subsequently an intramolecular elimination, unmasking the phosphate moiety. This type of pro-nucleotides does not exhibit any particular antiviral activity in comparison to the others.
Cyclosaligenyl nucleoside phosphotriesters—cyclo-Sal pro-nucleotides [Meier, C., Balzarini, J., Antiviral Res., 2006, 71, 282-292] have the formula (V)
Cyclo-Sal pro-nucleotides under in vitro cell culture conditions and, presumably, under in vivo physiological conditions as well, form nucleoside phosphates of type (II) by way of chemical hydrolysis exclusively. Hydrolysis of cyclic phosphotriester of a formula (V) is regiospecific towards an aryl phosphoester bond cleavage. The generated benzyl phosphoester is transformed into the final nucleoside 5′-phosphate of type (II), also by way of chemical hydrolysis. The investigated cyclo-Sal pro-nucleotides were directed towards HIV inhibition (AZT, ddA, d4G, d4T derivatives) and also towards hampering of HSV propagation (ACV derivatives). In the case of HIV inhibition the best results were observed for a d4T cyclo-Sal derivative.
Diaryl nucleoside phosphotriesters having the formula (VI)
are diaryl pro-nucleotides [Kraszewski A., et al., Bioorg. Med. Chem., 2009, 17, 3489-3498]. The concept of action of pro-nucleotides of type (VI) is based on a selection of the aryl phosphoester groups securing their appropriate susceptibility to chemical hydrolysis under physiological conditions. The aryl nucleoside phosphodiesters formed are good substrates for phosphoesterases, which hydrolyze them to the desired nucleoside 5′-phosphates of type (II). With a proper choice of phosphoester aryl groups the diaryl phosphotriesters of type (VI) are pro-nucleotides with a high anti-HIV activity.
Phosphoramidate diesters have the formulas (VII) and (VIII) [Cahard D., Mini-Rev. Med. Chem., 2004, 4, 371-381]
Phosphoramidate diesters of type (VII) and (VIII), amino acid derivatives, are structurally distinctive pro-nucleotides with the amide group formed by an amino acid bound to the phosphorus atom through the nitrogen atom of the α-amino group. Thus, it is an unequivocally defined group of pro-nucleotides. It is proven that phosphoramidate diesters of type (VII) and (VIII) act as pro-nucleotides. Anti-HIV activity of these compounds is tightly correlated with the kind of nucleoside (Nu), the structure and the kind of amino acid residue (R1, R2, and R3) and the aryl moiety (Ar). For example, for AZT derivative of type (VII) the best results were obtained for Ar=phenyl and tryptophan as the amino acid moiety, for which the antiviral activity was similar to that of AZT, while the cytotoxicity was significantly lower.
The pro-nucleotides of type (IX) are derivatives of other amines [Jochum A., et al., J. Organometal Chem., 2005, 690, 2614-2625]
It was proved that antiviral pro-nucleotides do not have to be amino acid derivatives. The investigated derivatives of simple amines exhibited the same or better pharmacokinetic parameters than those of analogous amino acid derivatives. It is assumed that in the compounds of type (IX) the SATE group is eliminated as the first one yielding phosphoramidate monoester that in turn is converted into nucleoside 5′-phosphate of type (II) in enzyme-catalyzed reactions. The compounds of type (IX) act as pro-nucleotides and show the anti-HIV activity higher or comparable to their parent nucleosides, while their toxicity is significantly lower.
Nucleoside bis(phosphoramidates) are pro-nucleotides having the formula (X) [Shipitsyn A. V., Nucleos. Nucleot. & Nucleic Acids, 2003, 22(5-8), 963-966]
The anti-HIV activity of nucleoside bis(phosphoramidates) of type (X) was investigated. The activity and cytotoxicity of these compounds was correlated tightly with the kind of nucleoside residue (AZT and d4T) and the substituents of the nitrogen atoms of the phosphordiamidate residues. Nucleoside 5′-phosphate of type (H) is formed from the compounds of type (X) in a stepwise chemical or enzyme-aided hydrolysis, the mechanism of which is likely, however still assumed only.
Nucleoside phosphoramidates are pro-nucleotides having the formula (XI) [Wagner C. R., et al., Mini-Rev. Med. Chem., 2004, 4, 409-419]
All pro-nucleotides of type (XI) described so far are nucleoside phosphoramidate derivatives of amino acids. It was proven that these compounds enter the cells as such and are subsequently transformed into the respective nucleoside 5′-phosphate of type (II) with the aid of phosphoamidase enzymes present in the cells. A number of pro-nucleotides of type (XI) are described, namely the derivatives of several nucleoside analogues of known antiviral (AZT, d4T, ddC, 3TC, ddA) and anticancer (5-fluoro-2′-deoxyuridine) activity. They display high antiviral activity and low toxicity. These parameters point to the compounds of type (XI) as important potential antiviral and anticancer therapeutics.
The only commercially available nucleotide drug used in AIDS therapy is the tenofovir having the formula (XII)
It is a substituted C-phosphonate of an acyclic purine nucleoside, the assumed mode of action of which classifies it as a drug of the pro-nucleotide type. Its structure resembles the POM compounds (the type (I) pro-nucleotides) and most likely it acts in the same manner, i.e. the decomposition of carbonate ester in the side chain releases the phosphonate residue and facilitates its further conversion into triphosphate, which is able to inhibit HIV replication.
In US 2001031745 patent (published on 2001, Oct., 18) a number of chemical compounds comprising nucleoside phosphoramidates, their preparation and therapeutic use in treating viral infections, particularly HIV and HBV, were disclosed. These compounds contain a substituted adenine analogue moiety—2-amino-6-(cyclopropylamino)-9H-purin-9-yl. The compounds display antiviral activity and are stable in acid environment. The application comprises also salts and esters of the phosphoramidates. A representative compound of the application is (1S, 4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-l-methylphenyl(methoxy-L-alaninyl)phosphoramidate.
In CN 1290707 patent (published on 2001, Apr., 11) a nucleoside 5′-thiophosphoryl amino acid ester compound, its preparation and application was disclosed. The compound was prepared through the processes of dissolving thiophosphoryl trichloride in dried tetrahydrofuran, addition of carbamate hydrochloride through stirring, dripping acid binding agent to react, filtering, rotating distillation to eliminate solvent and other low boiling point matters, hydrolysis in ammonia water and final silica gel chromatography. The compound of the invention exhibited antiviral, antitumor and HIV resisting activity and may have medical applications.
The US 2007042988 patent (published on 2007, Feb., 22) novel nucleotide compounds were disclosed, being phosphoramidate mono- and diesters prepared from modified nucleotides bonded with variously substituted amino acids and their analogues. The compounds were useful for the treatment of Hepatitis C Virus (HCV) mediated diseases. The invention further provided methods for treatment or prophylaxis of HCV mediated diseases with disclosed compounds and pharmaceutical compositions comprising these compounds.
In WO 2004113360 patent (published on 2004, Dec., 29) novel biologically active derivatives of 3′-azido-3′-deoxythymidine and 2′,3′-dideoxy-2′,3′-didehydrothymidine 5′-phosphates were disclosed as well as their application as antiviral agents, primarily against HIV. The invention makes it possible to develop and use novel compounds which are resistant to the action of phosphatases, capable to penetrate inside a cell and exhibit selective activity for suppressing cDNA biosynthesis which is catalyzed by the HIV reverse transcriptase. The disclosed compounds, i.e. phosphoramidates of nucleoside analogues comprising 5′-dimorpholinephosphordiamidate of 2′,3′-dideoxy-2′,3′-didehydrothymidine and 5′-dimorpholinephosphordiamidate of 3′-azido-3′-deoxythymidine, inhibit HIV replication and have defined formulas.
In US 2008249066 patent (published on 2008, Oct., 9) compounds useful in the treatment of viral diseases, compositions comprising them and methods of using them, are described. The compounds of the invention comprise a nucleoside or nucleoside analog linked usually through a phosphate group to one of selected groups of a lipid residue. The compounds described in the patent can be used for treating HIV infection, AIDS and other viral infections.
In WO 2008087558 patent (published on 2008, Jul., 24) compounds useful as antiviral or antitumor agents are presented. The compounds comprise nucleotide analogues that comprise tetrahydrofuranyl or tetrahydrothienyl moieties with quaternary centres at the 3′ position. The nucleotide analogues can be used to inhibit cancer or viruses. The invention also describes pharmaceutical formulations comprising the compounds designed to treat, prevent, or inhibit the diseases or conditions associated with cancers and viruses.
Despite numerous known solutions exploiting nucleoside derivatives as compounds for treating viral diseases, there is still a continuous need for an efficient solution allowing preparation of pharmaceutical compositions comprising water-soluble compounds showing the same or higher activity as the starting nucleoside and displaying simultaneously low toxicity.
The aim of the current invention is to find efficient compounds being a novel group of nucleotide derivatives, the physicochemical properties and anti-HIV pharmacokinetic parameters of which would be better than those of the parent nucleoside analogues. The invention applies to compounds that would be pro-nucleotides without chiral centres that could affect the transformation of the pro-nucleotide into the respective nucleosid-5′-yl phosphate.
The aims defined and the solutions to the problems associated with their realization, are achieved in this invention.